Throughout this application various publications are referred to in parenthesis or by reference number. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Cells recognize and respond to environmental stimuli via activation of intracellular biochemical pathways primarily comprised of protein kinases. These enzymes catalyze the phosphorylation of serine, threonine, and/or tyrosine residues on protein substrates. More specifically, protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl moieties of serine, threonine, and tyrosine. This deceptively modest reaction serves as a cornerstone for the extraordinarily complex phenomenon known as signal transduction, the biochemical process by which information is transmitted from the cell membrane to the cytoplasm and cell nucleus.62 For example, the binding event between growth factor and its receptor on the cell surface is signaled to the nucleus via protein kinase-mediated pathways. In response to this signal, genes are transcribed and the cell prepares itself for division.36 Mitosis is subsequently driven by a fine choreography of temporally- and spatially-regulated signaling pathways that ensure the myriad of biochemical processes required for replication occur in their proper chronological order. In short, signal transduction serves as a biochemical mechanism that drives an extraordinary array of biological phenomena. However, it would be simplistic to view signaling pathways as the molecular equivalent of the interstate highway system. The latter is fixed both in time and space. By contrast, kinase-mediated pathways not only evolved to rapidly form in response to some environmental stimulus, but their role in cellular homeostasis is dependent upon their rapid disassembly once the environmental signal has been acknowledged. Furthermore, the nature of the cellular response is dependent upon when and/or where a specific pathway is activated as well as by what other pathways may be simultaneously operating.
Protein kinases participate in the pathways that drive a variety of other important processes including apoptosis37. Members of this large enzyme family have been the objects of intense scientific scrutiny due to their role in disease onset and progression. The potential number of protein kinases encoded by the mammalian genome has been estimated to exceed 1,000.63 The protein kinase C (PKC) family of enzymes has been implicated in a wide variety of processes, including control of gene expression,72 mitotic progression,26-30,78 angiogenesis, carcinogenesis, metastasis, and insulin action.73 Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) plays a key role in the signaling pathways responsible for memory and learning.54,55 Protein kinases, and the signal transduction pathways in which they participate, are now recognized to be medicinally attractive targets of opportunity.1-4,74-76 Inhibitors of the protein kinase family not only hold great promise as therapeutic agents, but are also of profound utility in the characterization of signaling pathways.5 Consequently, there has been widespread interest in developing sensors of protein kinase activity, species that could furnish a visual readout of both where and when specific intracellular kinases are activated in response to a stimulus.
The substrate specificity of any given protein kinase is typically defined as the preferred amino acid sequence that envelops the serine, threonine, or tyrosine residue phosphorylated by the enzyme (consensus recognition sequences).53 In addition, protein kinases are typically divided into two families on the basis of their active site specificity: those that phosphorylate the aromatic phenol of tyrosine and those that catalyze the phosphorylation of the aliphatic alcohols of serine and threonine. PKC, PKG, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) share a strong sequence homology, and all three comprise what is commonly referred to as the “ACG” subfamily of protein kinases. Not surprising, these enzymes display overlapping sequence specificities with respect to both substrate and inhibitor peptides. However, their active site specificities are remarkably different.48,56 For example, whereas PKA is unable to phosphorylate alcohol-bearing residues that possess an α-stereocenter corresponding to that present in D-amino acids, both PKC and PKG readily phosphorylate residues containing this configuration.48,56,57 Furthermore, the differences in active site specificity between these otherwise closely related protein kinases are not just limited to stereochemical biases. For example, PKC phosphorylates meta- and para-substituted phenols, whereas PKA and PKG do not.22 Protein microarrays have been used to investigate essentially all of the protein kinases encoded by the yeast genome.77 
A variety of approaches have been described to assess protein kinase activity, including using phosphorylation-specific antibodies6,65,66 and cytoplasmic sampling with capillary electrophoresis.38 Ng et al. reported the detection of phosphorylated (activated) PKCα via fluorescence resonance energy transfer (FRET) using cyanine-labeled anti-phosphoPKCα and antiphosphoThr250 antibodies in fixed cells.6 In this particular case, the activity of PKCα activity is not directly measured, but is inferred by detecting a functional state of the enzyme. Nagai et al. described the imaging of PKA activity in cells expressing a protein composed of two green fluorescent protein (GFP) variants tethered by a PKA phosphorylation site.7 Phosphorylation of this protein generates a 23% decrease in FRET between the two GFPs. More recently, changes in FRET of 20-35% and 25-50% have been reported using genetically encoded reporters of protein tyrosine kinase69 and PKA68 activities, respectively.
Other studies have used peptide substrates that possess an appended fluorophore positioned near the site of phosphorylation8-10,51,70,71 The phosphorylation-induced change in fluorescence intensity in these systems is modest (<20%)8-10 and, as a consequence, the use of these substrates has often been limited to in vitro experiments with purified kinases. Nonetheless, peptide substrates possess a number of inherent advantages, including ready synthetic availability, straightforward modification with the wide array of commercially available fluorophores, and the potential for complete temporal and spatial control over both when and where the substrate is phosphorylated.11-17 Accordingly, there has been a need for fluorescently-labeled peptide substrates for protein kinases which undergo large changes in fluorescent intensity upon phosphorylation and which are suitable for in vitro and in vivo applications.